Homeodomains are a class of DNA-binding protein domains which have important roles in control of transcription and development in eukaryotes, and in some cases are involved in human oncogenesis. We are using the MAT a2 homeodomain of yeast (Li et al., 1995, Science 270, 262-269) as a model system to characterize the thermodynamics of protein folding and sequence-specific DNA binding by this protein motif. Using differential scanning and isothermal titration calorimetry, we measured the enthalpy, entropy, heat capacity, and Gibbs free energy changes of these processes for the wild-type sequence of a2 homeodomain (Carra & Privalov, 1997,Biochemistry 36, 526-535). The protein-DNA interaction is enthalpically driven at physiological temperatures. Protein folding and DNA binding are linked, as for several other DNA binding proteins. A comparison of the circular dichroism spectra of the free and DNA-bound protein species revealed that formation of protein structure is induced by DNA binding. The energies measured for association therefore include a component due to folding. We are currently extending these studies to mutant versions of the a2 homeodomain. Site-directed mutagenesis was used to create versions of the homeodomain that contain alanine substitutions in the DNA recognition helix, removing important contacts to DNA. Changes in the core packing of the protein were also made to assay the importance of certain conserved residues to folding, and the ability of the protein core to accept compensating substitutions. Deletion of the N-terminal arm of the protein, which wraps around the back of the DNA in the complex, results in a drastic loss of DNA binding affinity. The MATa2 homeodomain can also bind DNA cooperatively with the MATa1 homeodomain, as a heterodimer. This protein-protein interaction, and DNA binding by the heterodimer, will be studied using the isolated homeodomains and a genetically produced fusion of the two.